Total ammonia nitrogen inhibits medium-chain fatty acid biosynthesis by disrupting hydrolysis, acidification, chain elongation, substrate transmembrane transport and ATP synthesis processes

[Display omitted] •MCFA synthesis was inhibited at high total ammonia nitrogen (TAN) concentrations.•Hydrolysis, acidification and chain elongation processes were inhibited.•TAN stress did not result in a decrease in the abundance of CE bacteria.•The lower MCFA yield was due to the lower activities of key enzymes.•FAB pathway was the primary contributor for MCFA synthesis under TAN stress. This study used 16S rRNA gene sequencing and metatranscriptomic analysis to comprehensively illustrate how ammonia stress influenced medium-chain fatty acids (MCFA) biosynthesis. MCFA synthesis was inhibited at total ammonia nitrogen (TAN) concentrations above 1000 mg N/L. TAN stress hindered organic hydrolysis, acidification, and volatile fatty acids elongation. Chain-elongating bacteria (e.g., Clostridium_sensu_stricto_12, Clostridium_sensu_stricto_1, Caproiciproducens) abundance remained unchanged, but their activity decreased, partially due to the increased reactive oxygen species. Metatranscriptomic analysis revealed reduced activity of enzymes critical for MCFA production under TAN stress. Fatty acid biosynthesis pathway rather than reverse β-oxidation pathway primarily contributed to MCFA production, and was inhibited under TAN stress. Functional populations likely survived TAN stress through osmoprotectant generation and potassium uptake regulation to maintain osmotic pressure, with NADH-ubiquinone oxidoreductase potentially compensating for ATP loss. This study enhances understanding of MCFA biosynthesis under TAN stress, aiding MCFA production system stability and efficiency improvement.